U.S. patent number 7,205,336 [Application Number 10/512,507] was granted by the patent office on 2007-04-17 for .beta.-secretase inhibitors.
This patent grant is currently assigned to Merck & Co., Inc.. Invention is credited to Stephen F. Brady, Ming-Chih Crouthamel, Ming-Tain Lai.
United States Patent |
7,205,336 |
Lai , et al. |
April 17, 2007 |
.beta.-secretase inhibitors
Abstract
The present invention provides compounds that are inhibitors of
the proteolytic activity of the enzyme .beta.-secretase,
pharmaceutically acceptable salts of the compounds, pharmaceutical
compositions comprising the compounds, processes for making the
compounds, and methods of using the compounds to treat Alzheimers
disease.
Inventors: |
Lai; Ming-Tain (Lansdale,
PA), Crouthamel; Ming-Chih (Perkasie, PA), Brady; Stephen
F. (Philadelphia, PA) |
Assignee: |
Merck & Co., Inc. (Rahway,
NJ)
|
Family
ID: |
29584318 |
Appl.
No.: |
10/512,507 |
Filed: |
May 14, 2003 |
PCT
Filed: |
May 14, 2003 |
PCT No.: |
PCT/US03/15109 |
371(c)(1),(2),(4) Date: |
October 26, 2004 |
PCT
Pub. No.: |
WO03/099202 |
PCT
Pub. Date: |
December 04, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050164953 A1 |
Jul 28, 2005 |
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Current U.S.
Class: |
514/563;
562/448 |
Current CPC
Class: |
C07C
237/22 (20130101); C07D 317/70 (20130101); C07C
2601/04 (20170501) |
Current International
Class: |
A61K
31/195 (20060101); C07C 229/00 (20060101) |
Foreign Patent Documents
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|
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WO 89/04833 |
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Jun 1989 |
|
WO |
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WO 01/00665 |
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Jan 2001 |
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WO |
|
Primary Examiner: Zucker; Paul A.
Attorney, Agent or Firm: Todaro; John C. Winokur; Melvin
Claims
What is claimed is:
1. A compound of Formula 1 or a pharmaceutically acceptable salt
thereof: ##STR00014## wherein: R.sub.1 is independently selected
from the group consisting of: C.sub.1-4 alkyl, cyclic alkyl,
arylalkyl, and aryloxyalkyl; R.sub.2 is independently selected from
the group consisting of: hydrogen, ##STR00015## R.sub.3 is
independently selected from the group consisting of: C.sub.1-4
alkyl, cycloalkyl, and arylalkyl; and R.sub.4 is independently
selected from the group consisting of: C.sub.1-4 alkyl and
arylalkyl.
2. The compound of claim 1 wherein: R.sub.1 is independently
selected from the group consisting of: ##STR00016##
3. The compound of claim 1 wherein; R.sub.3 is independently
selected from the group consisting of: ##STR00017##
4. The compound of claim 1 wherein: R.sub.4 is independently
selected from the group consisting of: ##STR00018##
5. The compound of claim 1 wherein: R.sub.1 is independently
selected from the group consisting of: ##STR00019## R.sub.3 is
independently selected from the group consisting of: ##STR00020##
and R.sub.4 is independently selected from the group consisting of:
##STR00021##
6. A compound of Formula 2 or a pharmaceutically acceptable salt
thereof: ##STR00022## wherein: R.sub.1 is independently selected
from the group consisting of: C.sub.1-4 alkyl, cyclic alkyl,
arylalkyl, and aryloxyalkyl; R.sub.2 is independently selected from
the group consisting of: hydrogen, ##STR00023## R.sub.3 is
independently selected from the group consisting of: C.sub.1-4
alkyl, cycloalkyl, and arylalkyl; and R.sub.4 is independently
selected from the group consisting of: C.sub.1-4 alkyl and
arylalkyl.
7. The compound of claim 6 wherein: R.sub.1 is independently
selected from the group consisting of: ##STR00024##
8. The compound of claim 6 wherein: R.sub.3 is independently
selected from the group consisting of: ##STR00025##
9. The compound of claim 6 wherein: R.sub.4 is independently
selected from the group consisting of: ##STR00026##
10. The compound or pharmaceutically acceptable salt thereof of
claim 6 wherein: R.sub.1 is independently selected from the group
consisting of: ##STR00027## R.sub.3 is independently selected from
the group consisting of: ##STR00028## and R.sub.4 is independently
selected from the group consisting of: ##STR00029##
11. A compound which is selected from the group consisting of:
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## or
a pharmaceutically acceptable salt thereof.
12. A pharmaceutical composition comprising a compound of claim 1
or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier.
13. A method for the treatment of a person suffering from or prone
to Alzheimer's disease comprising administering to that person an
effective amount of a compound of claim 1 or a pharmaceutically
acceptable salt thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. .sctn. 119(e) from
Provisional Application No. 60/381,535, filed May 17, 2002.
BACKGROUND OF THE INVENTION
Alzheimer's disease is a common, chronic neurodegenerative disease,
characterized by a progressive loss of memory and sometimes severe
behavioral abnormalities, as well as an impairment of other
cognitive functions that often leads to dementia and death. It
ranks as the fourth leading cause of death in industrialized
societies after heart disease, cancer, and stroke. The incidence of
Alzheimer's disease is high, with an estimated 2.5 to 4 million
patients affected in the United States and perhaps 17 to 25 million
worldwide. Moreover, the number of sufferers is expected to grow as
the population ages.
A characteristic feature of Alzheimer's disease is the presence of
large numbers of insoluble deposits, known as amyloid plaques, in
the brains of those affected. Autopsies have shown that amyloid
plaques are found in the brains of virtually all Alzheimer's
patients and that the degree of amyloid plaque deposition
correlates with the degree of dementia (Cummings & Cotman,
1995, Lancet 326:1524 1587). While some opinion holds that amyloid
plaques are a late stage by-product of the disease process, the
consensus view is that amyloid plaques are more likely to be
intimately, and perhaps causally, involved in Alzheimer's
disease.
A variety of experimental evidence supports this view. For example,
A.beta., a primary component of amyloid plaques, is toxic to
neurons in culture and transgenic mice that overproduce A.beta. in
their brains show significant deposition of A.beta. into amyloid
plaques as well as significant neuronal toxicity (Yankner, 1990,
Science 250:279 282; Mattson et al., 1992, J. Neurosci. 12:379 389;
Games et al., 1995, Nature 373:523 527; LaFerla et al., 1995,
Nature Genetics 9:21 29). Mutations in the Alzheimer's precursor
protein (APP) gene, leading to increased A.beta. production, have
been linked to heritable forms of Alzheimer's disease (Goate et
al., 1991, Nature 349:704 706; Chartier-Harlan et al., 1991, Nature
353:844 846; Murrel et al., 1991, Science 254:97 99; Mullan et al.,
1992, Nature Genetics 1:345 347). Injection of the insoluble,
fibrillar form of A.beta. into monkey brains results in the
development of pathology (neuronal destruction, tau
phosphorylation, microglial proliferation) that closely mimics
Alzheimer's disease in humans (Geula et al., 1998, Nature Medicine
4:827 831). See Selkoe, 1994, J. Neuropathol. Exp. Neurol. 53:438
447 for a review of the evidence that A.beta. and amyloid plaques
have a central role in Alzheimer's disease.
A.beta., a 39 43 amino acid peptide derived by proteolytic cleavage
of APP, is the major component of amyloid plaques (Glenner &
Wong, 1984, Biochem. Biophys. Res. Comm. 120:885 890). APP is
actually a family of polypeptides produced by alternative splicing
from a single gene. Major forms of APP are known as APP.sub.695,
APP.sub.751, and APP.sub.770, with the subscripts referring to the
number of amino acids in each splice variant (Ponte et al., 1988,
Nature 331:525 527; Tanzi et al., 1988, Nature 331:528 530;
Kitaguchi et al., 1988, Nature 331:530 532). APP is membrane bound
and undergoes proteolytic cleavage by at least two pathways. In one
pathway, cleavage by an enzyme known as .alpha.-secretase occurs
while APP is still in the trans-Golgi secretory compartment
(Kuentzel et al., 1993, Biochem J. 295:367 378). This cleavage by
.alpha.-secretase occurs within the A.beta. portion of APP, thus
precluding the formation of A.beta.. In another proteolytic
pathway, cleavage of the Met.sub.671-Asp.sub.672 bond (numbered
according to the 751 amino acid protein) by an enzyme known as
.beta.-secretase occurs. This cleavage by .beta.-secretase
generates the N-terminus of A.beta.. The C-terminus is formed by
cleavage by a second enzyme known as .gamma.-secretase. The
C-terminus is actually a heterogeneous collection of cleavage sites
rather than a single site since .gamma.-secretase activity occurs
over a short stretch of APP amino acids rather than at a single
peptide bond. Peptides of 40 or 42 amino acids in length (A.beta.40
and A.beta.42, respectively) predominate among the C-termini
generated by .gamma.-secretase. A.beta.42 is more prone to
aggregation than A.beta.40, is the major component of amyloid
plaque (Jarrett et al., 1993, Biochemistry 32:4693 4697; Kuo et
al., 1996, J. Biol. Chem. 271:4077 4081), and its production is
closely associated with the development of Alzheimer's disease
(Sinha & Lieberburg, 1999, Proc. Natl. Acad. Sci. USA 96:11049
11053). The bond cleaved by .gamma.-secretase appears to be
situated within a transmembrane domain of APP. It is unclear as to
whether the C-termini of A.beta.40 and A.beta.42 are generated by a
single .gamma.-secretase protease with sloppy specificity or by two
distinct proteases. For a review that discusses APP and its
processing, see Selkoe, 1998, Trends Cell. Biol. 8:447 453.
Much interest has focused on the possibility of inhibiting the
development of amyloid plaques as a means of preventing or
ameliorating the symptoms of Alzheimer's disease. To that end, a
promising strategy is to inhibit the activity of .beta.- and
.gamma.-secretase, the two enzymes that together are responsible
for producing A.beta.. This strategy is attractive because, if the
formation of amyloid plaques as a result of the deposition of
A.beta. is a cause of Alzheimer's disease, inhibiting the activity
of one or both of the two secretases would intervene in the disease
process at an early stage, before late-stage events such as
inflammation or apoptosis occur. Such early stage intervention is
expected to be particularly beneficial (see, e.g., Citron, 2000,
Molecular Medicine Today 6:392 397).
Various groups have cloned and sequenced cDNA encoding a protein
that is believed to be .beta.-secretase (Vassar et al., 1999,
Science 286:735 741; Hussain et al., 1999, Mol. Cell. Neurosci.
14:419 427; Yan et al., 1999, Nature 402:533 537; Sinha et al.,
1999, Nature 402:537 540; Lin et al., 2000, Proc. Natl. Acad. Sci.
USA 97:1456 1460). Hong et al., 2000, Science 290:150 153
determined the crystal structure of the protease domain of human
.beta.-secretase complexed with an eight-residue peptide-like
inhibitor at 1.9 angstrom resolution. Compared to other human,
aspartic proteases, the active site of human .beta.-secretase is
more open and less hydrophobic, contributing to the broad substrate
specificity of human .beta.-secretase (Lin et al., 2000, Proc.
Natl. Acad. Sci. USA 97:1456 1460).
Ghosh et al., 2000, J. Am. Chem. Soc. 122:3522 3523 disclosed two
inhibitors of .beta.-secretase, OM99-1 and OM99-2, that are
modified peptides based on the .beta.-secretase cleavage site of
the Swedish mutation of APP (SEVNL/DAEFR, with "/" indicating the
site of cleavage). OM99-1 has the structure VNL*AAEF (with "L*A"
indicating the uncleavable hydroxyethylene transition-state
isostere of the LA peptide bond) and exhibits a K.sub.i towards
recombinant .beta.-secretase produced in E. coli of
6.84.times.10.sup.-8 M.+-.2.72.times.10.sup.-9 M. OM99-2 has the
structure EVNL*AAEF (with "L*A" indicating the uncleavable
hydroxyethylene transition-state isostere of the LA peptide bond)
and exhibits a K.sub.i towards recombinant .beta.-secretase
produced in E. coli of 9.58.times.10.sup.-9
M.+-.2.86.times.10.sup.-10 M. OM99-1 and OM99-2, as well as related
compounds, are described in International Patent Publication WO
01/00665.
SUMMARY OF THE INVENTION
The present invention is directed to compounds that are aspartyl
protease inhibitors capable of inhibiting the cleavage of APP by
.beta.-secretase. The compounds of the present invention are useful
in the treatment of Alzheimer's disease. Pharmaceutical compounds
containing the compounds and methods of using the compounds to
treat Alzheimer's disease are disclosed.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds that are inhibitors of the
proteolytic activity of the enzyme .beta.-secretase,
pharmaceutically acceptable salts of the compounds, pharmaceutical
compositions comprising the compounds, processes for making the
compounds, and methods of using the compounds to treat Alzheimer's
disease.
The compounds of the present invention are useful for treating
Alzheimer's disease by inhibiting the activity of .beta.-secretase,
thereby preventing the formation of A.beta., thus leading to a
lessening of the deposition of plaques in the brains of those
treated with the compounds and a lessening of other deleterious
effects of A.beta..
The present invention, in one aspect, provides a compound having
the following structure (Formula 1):
##STR00001##
The present invention also provides compounds in which the carbon
to which the R.sub.2 group is attached is in the R configuration
(Formula 2):
##STR00002##
In Formula 1 and Formula 2, R.sub.1 is independently selected from
the group consisting of: C.sub.1-4 alkyl, cyclic alkyl, arylalkyl,
and aryloxyalkyl.
In certain embodiments, R.sub.1 is independently selected from the
group consisting of:
##STR00003##
In Formula 1 and Formula 2, R.sub.2 is independently selected from
the group consisting of: hydrogen, C.sub.1-4 alkyl, and substituted
C.sub.1-4 alkyl.
In certain embodiments, R.sub.2 is independently selected from the
group consisting of: hydrogen,
##STR00004##
In Formula 1 and Formula 2, R.sub.3 is independently selected from
the group consisting of: C.sub.1-4 alkyl, cyclic alkyl, and
arylalkyl.
In certain embodiments, R.sub.3 is independently selected from the
group consisting of:
##STR00005##
Compounds of Formula 1 can generally be used as .beta.-secretase
inhibitors as racemic mixtures of the R and S isomers at the carbon
atom to which the R.sub.2 group is attached in Formula 1 although
it has been found that the R isomer is the active isomer.
Therefore, the compounds of Formula 1 are preferably used as pure
preparations of the R isomers.
The term "C.sub.1-4 alkyl" refers to a saturated monovalent
hydrocarbon radical, linear or branched, having from 1 4 carbons.
Examples are: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,
t-butyl.
The term "substituted C.sub.1-4 alkyl" refers to a C.sub.1-4 alkyl
substituted with one or more substituents selected from the group
consisting of: hydroxyl, acetyloxy, methyloxy, and ethyloxy.
The term "cyclic alkyl" refers to a saturated cyclic hydrocarbon
radical having from 4 7 carbons. Preferably, the cyclic alkyl is
cyclobutyl, methylenecyclobutyl, cyclopentyl, methylenecyclopentyl,
methylenecyclohexyl, cyclohexyl, methylenecycloheptyl, or
cycloheptyl.
The term "arylalkyl" refers to an aromatic group selected from
phenyl and naphthyl attached to an alkyl group, which alkyl group
is in turn attached to Formula 1 or Formula 2 at the R.sub.1 or
R.sub.3 position. The alkyl group may be methylene, ethylene,
propylene, n-butylene, or sec-butylene.
The term "aryloxyalkyl" refers to aromatic groups selected from
phenyl and naphthyl attached through an oxygen atom to an alkyl
group, which alkyl group is in turn attached to Formula 1 or
Formula 2 at the R.sub.1 position. When the aromatic group includes
a fused ring, the link between the fused ring and the oxygen may be
at any point along the fused ring. The alkyl group may be
methylene, ethylene, propylene, n-butylene, or sec-butylene.
The present invention provides compounds having the following
structures:
##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011## ##STR00012##
The potency of the above-disclosed inhibitors was tested in the
FRET assay described in Example 1. The IC.sub.50 (.mu.M) values
ranged from 0.4 to 70.
The present invention includes pharmaceutical compositions
comprising one or more compounds of this invention. The compounds
of the invention are generally combined with pharmaceutically
acceptable carriers to form pharmaceutical compositions. Examples
of such carriers and methods of formulation of pharmaceutical
compositions containing compounds of the invention and carriers can
be found in Gennaro, ed., Remington's Pharmaceutical Sciences,
18.sup.th Edition, 1990, Mack Publishing Co., Easton, Pa.
Pharmaceutical carriers can include, e.g., conventional tableting
ingredients such as corn starch, lacose, sucrose, sorbitol, talc,
stearic acid, magnesium stearate, dicalcium phosphate or gums or
surfactants such as sorbitan monooleate, polyethylene glycol, and
other pharmaceutical diluents, e.g., water, to form a solid
preformulation composition containing a homogeneous mixture of a
compound of the invention, or a pharmaceutically acceptable salt
thereof. To form a pharmaceutically acceptable composition suitable
for effective administration, such compositions will contain a
therapeutically effective amount of the compounds of the
invention.
Therapeutic or prophylactic compositions are administered to an
individual in amounts sufficient to treat or prevent Alzheimer's
disease or to alleviate the symptoms of Alzheimer's disease. The
effective amount can vary according to a variety of factors such as
the individual's condition, weight, gender, and age. Other factors
include the mode of administration. The appropriate amount can be
determined by a skilled physician. Generally, an effective amount
will be from about 0.01 to about 1,000, preferably from about 0.1
to about 250, and even more preferably from about 1 to about 50 mg
per adult human per day.
Compositions can be used alone at appropriate dosages.
Alternatively, co-administration or sequential administration of
other agents can be desirable.
The compositions can be administered in a wide variety of
therapeutic dosage forms in conventional vehicles for
administration. For example, the compositions can be administered
in such oral dosage forms as tablets, capsules (each including
timed release and sustained release formulations), pills, powders,
granules, elixirs, tinctures, solutions, suspensions, syrups and
emulsions, or by injection. Likewise, they can also be administered
in intravenous (both bolus and infusion), intraperitoneal,
subcutaneous, topical with or without occlusion, or intramuscular
form, all using forms well known to those of ordinary skill in the
pharmaceutical arts.
Compositions can be administered in a single daily dose, or the
total daily dosage can be administered in divided doses of two,
three, four or more times daily. Furthermore, compositions can be
administered in intranasal form via topical use of suitable
intranasal vehicles, or via transdermal routes, using those forms
of transdermal skin patches well known to those of ordinary skill
in that art. To be administered in the form of a transdermal
delivery system, the dosage administration will, of course, be
continuous rather than intermittent throughout the dosage
regimen.
The dosage regimen utilizing the compositions is selected in
accordance with a variety of factors including age, weight, sex,
and medical condition of the patient; the severity of the
Alzheimer's disease and other conditions the patient may suffer
from; the route of administration; the renal, hepatic, and
cardiovascular function of the patient; and the particular
composition thereof employed. A physician of ordinary skill can
readily determine and prescribe the effective amount of the
composition required to prevent, counter or arrest the progress of
the Alzheimer's disease. Optimal precision in achieving
concentrations of composition within the range that yields efficacy
without toxicity requires a regimen based on the kinetics of the
composition's availability to the brain. This involves a
consideration of the distribution, equilibrium, and elimination of
a composition as well as the composition's ability to cross the
blood/brain barrier.
Frequently, it will be desirable or necessary to introduce the
pharmaceutical compositions containing the compounds of the present
invention directly or indirectly to the brain. Direct techniques
usually involve placement of a drug delivery catheter into the
host's ventricular system to bypass the blood-brain barrier.
Indirect techniques, which are generally preferred, involve
formulating the compositions to provide for drug latentiation by
the conversion of hydrophilic drugs into lipid-soluble drugs.
Latentiation is generally achieved through blocking of the
hydroxyl, carboxyl, and primary amine groups present on the drug to
render the drug more lipid-soluble and amenable to transportation
across the blood-brain barrier. Alternatively, the delivery of
hydrophilic drugs can be enhanced by intra-arterial infusion of
hypertonic solutions which can transiently open the blood-brain
barrier.
The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable non-toxic bases or acids,
including inorganic or organic bases and inorganic or organic
acids. Salts derived from inorganic bases include aluminum,
ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,
manganic salts, manganous, potassium, sodium, zinc, and the like.
Particularly preferred are the ammonium, calcium, magnesium,
potassium, and sodium salts. Salts in the solid form may exist in
more than one crystal structure, and may also be in the form of
hydrates. Salts derived from pharmaceutically acceptable organic
non-toxic bases include salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines, and basic ion exchange resins,
such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylene-diamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine, and the like.
When the compound of the present invention is basic, salts may be
prepared from pharmaceutically acceptable non-toxic acids,
including inorganic and organic acids. Such acids include acetic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic,
fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic,
lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric,
pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,
p-toluenesulfonic acid, and the like. Particularly preferred are
citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric,
and tartaric acids.
The present invention further provides the use of a compounds of
the present invention or a pharmaceutically acceptable salt thereof
in the manufacture of a medicament for the treatment, prevention,
or alleviation of the symptoms of Alzheimer's disease.
Also disclosed is a method of treatment of a person suffering from
or prone to Alzheimer's disease which comprises administering to
that person an effective amount of a compound according to the
present invention or a pharmaceutically acceptable salt thereof
where said effective amount delays the appearance of, delays the
progression of, or alleviates the symptoms of Alzheimer's
disease.
The present invention further provides the use of a compounds of
the present invention or a pharmaceutically acceptable salt thereof
in the manufacture of a medicament for the treatment, prevention,
or alleviation of the symptoms of Down's syndrome, vascular
dementia, mild cognitive impairment, or hereditary cerebral
hemmorhage with amyloidosis of the Dutch type, as well as to
preventing or delaying the onset of Alzheimer's disease in those
who would progress from mild cognitive impairment to Alzheimer's
disease.
Also disclosed is a method of treatment of a person which comprises
administering to that person an effective amount of a compound
according to the present invention or a pharmaceutically acceptable
salt thereof where said effective amount delays the appearance of,
delays the progression of, or alleviates the symptoms of Down's
syndrome, vascular dementia, mild cognitive impairment, or
hereditary cerebral hemmorhage with amyloidosis of the Dutch
type.
The liquid forms in which the novel compositions of the present
invention may be incorporated for administration orally or by
injection include aqueous solutions, suitably flavored syrups,
aqueous or oil suspensions, and flavored emulsions with edible oils
such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as
well as elixirs and similar pharmaceutical vehicles. Suitable
dispersing or suspending agents for aqueous suspensions include
synthetic and natural gums such as tragacanth, acacia, alginate,
dextran, sodium carboxymethylcellulose, methylcellulose,
polyvinyl-pyrrolidone, or gelatin.
The compounds of the present invention have at least one asymmetric
center at the carbon atom marked with a star in Formula 3.
Additional asymmetric centers may be present depending upon the
nature of the various substituents on the compound. Each such
asymmetric center will independently produce two optical isomers
and it is intended that all of the possible optical isomers and
diastereomers in mixtures and as pure or partially purified
compounds are included within the ambit of this invention. The most
preferred compounds of this invention have the R configuration at
the carbon atom marked with a star in Formula 3.
Where the herein-described processes for the preparation of the
compounds according to the invention give rise to mixtures of
stereoisomers, it may be advantageous to use the racemic mixtures
without further purification of individual stereoisomers. For
example, the racemic mixture of the compound of Formula 3 is a
potent inhibitor even as a racemic mixture of the R and S isomers
at the carbon atom marked with a star in Formula 3. Alternatively,
it may be desirable to separate stereoisomers. This can be done by
conventional techniques such as preparative chromatography.
The absolute stereochemistry of compounds of the present invention
may be determined by x-ray crystallography of crystalline products
of the compounds or crystalline intermediates which may be
derivatized, if necessary, with a reagent containing an asymmetric
center of known absolute configuration.
During the synthetic sequences described herein, it may be
necessary and/or desirable to protect sensitive or reactive groups
on any of the molecules concerned. This may be achieved by means of
conventional protecting groups, such as those described in
Protective Groups in Organic Chemistry, ed. J. P. W. McOmie, Plenum
Press, 1973; and Protective Groups in Organic Synthesis, T. W.
Greene & P. G. M. Wuts, John Wiley & Sons, 1991.
In addition to the assays exemplified herein, a variety of assays
for measuring the ability of a substance to inhibit
.beta.-secretase activity are known in the art and can be used in
connection with the compounds of the present invention. Such assays
are described in, e.g., Turner et al., 2001, Biochem. 40:10001
10006; Steinhilb et al., 2001, J. Biol. Chem. 276:4476 4484. Where
purified .beta.-secretase is referred to herein, .beta.-secretase
is purified by the procedures described in Shi et al., 2001, J.
Biol. Chem. 276:10366 10373. The following non-limiting examples
are presented to illustrate the invention.
EXAMPLE 1
FRET Assay for Inhibition of .beta.-Secretase Activity
A. Reaction Mixture: (Plate=Costar #3915) volumes are given in
.mu.L
TABLE-US-00001 25 NH.sub.4OAc, 200 mM pH 4.5.0 10 BSA, 1 mg/ml
(Bovine Fraction V, Sigma #9647) 10 EDTA, 150 mM 2 10% CHAPS 2
Deferoxamine Mesylate, 50 mM (Sigma #D9533) 10 .beta.-secretase,
200 nM in H.sub.2O 31 H.sub.2O 8 Compound to be tested in DMSO 98
Total
1. The compound and .beta.-secretase are incubated at room
temperature for 30 min with shaking 2. Two microliters of 62.5
.mu.M FRET substrate is added into the solution and the resulting
mixtures are incubated at room temperature with shaking for 5 min
3. The kinetic reading on the LJL Analyst AD is taken every 5 min
fluorescence intensity; tamraqst-7; excitation 530 nm; emission 580
nm; dichroic mirror rhodamine 561; duration=300 sec, 5 reads) 4.
The K.sub.i of the inhibitors are determined by the equation of
((v.sub.0/v.sub.i)-1=[I]/K.sub.i). The v.sub.0 and v.sub.i
represent the initial rate of substrate hydrolysis in the absence
and presence of inhibitor (v.sub.0, v.sub.i), respectively. Note:
a. .beta.-secretase final concentration=30 nM (If using less, e.g.,
2 nM .beta.-secretase, change the reading duration to 3600 sec.) b.
DMSO=10% c. FRET 12mer final concentration=1.25 .mu.M d.
Hydrolysis<10% Reader: LJL Analyst AD Excitation: 530, FWHM 25
Emission: 580, FWHM 10 Dichroic 561
EXAMPLE 2
Additional FRET Assay for Inhibition of .beta.-Secretase
Activity
TABLE-US-00002 PART 1 25 .mu.L 4x NaOAc (200 mM, pH 4.5) 10 .mu.L 1
mg/ml BSA (Bovine Fraction V, Sigma #9647) 2 .mu.L 10% CHAPS 2
.mu.L 50 mM Deferroxamine Mesylate (Sigma #D9533) 10 .mu.L 150 mM
EDTA, pH 4.5 41 .mu.L dH.sub.2O 10 .mu.L two inhibitors at
different concentrations with fixed ratio transfer 80 .mu.L to part
2; black fluorescence 96-well plate (Costar #3915). PART 2 80 .mu.L
part 1 mixture 18 .mu.L .beta.-secretase mixture: 5 .mu.L 4x NaOAc,
4.5 2 .mu.L 1 mg/mL BSA (0.1%) 2 .mu.L 150 mM EDTA, 4.5 0.4 .mu.L
10% CHAPS 0.4 .mu.L 50 mM Def. Mes. 6.1 .mu.L dH.sub.2O 2 .mu.L 0.1
.mu.g/.mu.L .beta.-secretase (.beta.-secretase diluted in 20 mM
TRIS, 7.2; 125 mM NaCl)
incubate for 30 min at room temperature; shaking. add 2 .mu.L 125
.mu.M FRET substrate. Take a reading every 5 min on the LJL Analyst
AD for 20 min (fluorescence intensity; tamra-qst-7; excitation 530
nm; emission 580 nm; rhodamine 561 dichroic mirror). V.sub.0/vi
were calculated at each inhibitor concentration and plot against
the concentration of one of the inhibitors
EXAMPLE 3
Synthesis of the Parent Compound 3 of the Present Invention as the
Single Isomeric Constituent 25
##STR00013##
The compound of Formula 25 is the single diastereoisomer at the C-2
(*) corresponding to the isomer mixture 3 as per Formula 1. The
starting Fmoc derivative Ia is prepared using a published route
(Ghosh, A. K.; Dhin, D.; Downs, D.; Koelsch, G.; Lin, X.;
Ermolieff, J.; Tang, J. Design of Potent Inhibitors for Human Brain
Memapsin 2 (.beta.-Secretase). 2000, 122, 3522 3523), with the
adaptation that for introduction of the ethyl group at C-2 ethyl
iodide is employed in the stereoselective alkylation of the lactone
intermediate.
Thus, to a solution of 1.0 g (1.8 mmol) of intermediate Ia in 30 ml
of DMF is added 0.44 g (2.9 mmol) of HOBt, followed by 0.22 ml
(0.16 g, 2.2 mmol) of 2-methylpropylamine incrementally, making
sure the pH stays below 7.5 (moistened narrow-range indicator
paper). Then 0.56 g (2.9 mmol) of EDC is added, followed by
periodic N-methyl morpholine (NMM) to maintain pH 6 7. After 20 hr
water is added, the solvent is removed under reduced pressure, and
the residue is partitioned with H.sub.2O/EtOAc plus dil.
KHSO.sub.4, washing in turn with H.sub.2.sub.O, satd NaHCO.sub.3,
and 50% aq NaCl (2.times.). After drying (Na.sub.2SO.sub.4), the
EtOAc is removed under reduced pressure and the product is purified
by silica gel column chromatography, eluting with EtOAc/hexane to
isolate intermediate IIa as a colorless oil (LC-MS, NMR).
A solution of 115 mg (0.19 mmol) of intermediate IIa in 20 ml of
EtOAc is cooled to -20.degree. and saturated with HCl gas.
Saturation is maintained at a temperature of 0.degree. for 20 30
min, followed by purging of the solution with N.sub.2 for 1 2 hr.
The solvent is then removed under reduced pressure, and the residue
is dried under vacuum for 1 hr and dissolved in 6 ml of DMF,
followed by 3 ml of piperidine. After 10 min the solvent is removed
under reduced pressure, 20 ml of DMF is added, the DMF is removed
under vacuum, and the DMF addition/removal procedure is repeated
twice, followed by drying under high vacuum for 2 hr. To a solution
of this residue in 6 ml of DMF is added 73 mg (0.23 mmol) of
Boc-aspartic acid .alpha.-benzyl ester, followed by 43 mg (0.32
mmol) of HOBt and enough NMM to bring the pH to 7.5. Then 65 mg
(0.34 mmol) of EDC is introduced and the reaction mixture is
stirred for 20 hr, followed by addition of H.sub.2O and extractive
workup with EtOAc as above. Solvent removal under reduced pressure
and addition of ether affords 70 mg of intermediate IIIa as a white
solid (TLC, LC-MS).
A solution of intermediate IIIa in 20 ml of EtOAc is treated with
HCl gas exactly as described above, except saturation is maintained
for only 10 min before purging with N.sub.2. The residue after
drying under vacuum is dissolved in 6 ml of DMF, to which are added
29 mg (0.15 mmol) of 1-naphthyloxyacetic acid and 28 mg (0.21 mmol)
of HOBt, then sufficient NMM (approx. 0.05 ml) to bring the pH to
7.5. To the solution is added 41 mg (0.21 mmol) of EDC, followed by
stirring for 20 hr with addition of NMM as needed to maintain pH 7.
Workup proceeds as described above for the isolation of
intermediate IIIa, and the crude coupling product is dissolved in
25 ml of 95% EtOH and hydrogenated at atmospheric pressure (balloon
reservoir) with 36 mg of 10% Pd-on-carbon for 1 hr. After
filtration through Celite and concentration under reduced pressure,
the crude product is purified by preparative HPLC on a C18 column
using a gradient of 0.1% aq. NH.sub.4HCO.sub.3/CH.sub.3CN.
Lyophilization affords 30 mg of compound 25 (LC-MS, HPLC, N),
identical with the active diastereoisomer of the C-2 isomeric
mixture 3.
EXAMPLE 4
Synthesis of Compound 8 of the Present Invention
The starting Boc derivative Ib is prepared using a published route
(Ghosh, A. K.; Dhin, D.; Downs, D.; Koelsch, G.; Lin, X.;
Ermolieff, J.; Tang, J. Design of Potent Inhibitors for Human Brain
Memapsin 2 (.beta.-Secretase). 2000, 122, 3522 3523), with the
adaptation that Boc-phenylalanine is employed as the starting
material for incorporation of the benzyl group at C-5, and ethyl
iodide is employed in the stereoselective alkylation of the lactone
intermediate for introduction of the ethyl group at C-2.
Thus, to a solution of 0.19 g (0.41 mmol) of intermediate Ib in 6
ml of DMF is added 0.089 g (0.59 mmol) of HOBt, followed by 0.058
ml (0.042 g, 0.58 mmol) of 2-methylpropylamine. Then 0.12 g (0.62
mmol) of EDC is added, followed by incremental NMM to maintain pH
7.5. After 20 hr water is added, the solvent is removed under
reduced pressure, and the residue is partitioned with
H.sub.2O/EtOAc plus dil. KHSO.sub.4, washing in turn with H.sub.2O,
satd NaHCO.sub.3, and 50% aq NaCl (2.times.). After drying
(Na.sub.2SO.sub.4), the EtOAc is removed under reduced pressure,
followed by drying under vacuum to afford intermediate IIb as a
colorless oil (LC-MS).
A solution of 192 mg (0.37 mmol) of intermediate IIb in 20 ml of
EtOAc is cooled to -20.degree. and saturated with HCl gas.
Saturation is maintained at a temperature of 0.degree. for 30 min,
followed by purging of the solution with N.sub.2 for 1 2 hr. The
solvent is then removed under reduced pressure, and the residue is
dried under vacuum for 1 hr and dissolved in 9 ml of DMF, followed
by addition of 10 .mu.l of NMM. To this solution is added 159 mg
(0.49 mmol) of Boc-aspartic acid .alpha.-benzyl ester, followed by
106 mg (0.79 mmol) of HOBt and enough NMM to bring the pH to 7.5.
Then 146 mg (0.76 mmol) of EDC is introduced and the reaction
mixture is stirred for 20 hr, followed by addition of H.sub.2O and
extractive workup with EtOAc as above. Solvent removal under
reduced pressure and addition of ether affords 186 mg of
intermediate IIIb as a white solid (TLC, LC-MS).
A solution of intermediate IIIb in 30 ml of EtOAc is treated with
HCl gas exactly as described above, except saturation is maintained
for only 10 min before purging with N.sub.2. The residue after
drying under vacuum is dissolved in 12.0 ml of DMF, to which are
added 78 mg (0.51 mmol) of HOBt and sufficient NMM (approx. 0.045
ml) to bring the pH to 7.0. To 2.0 ml of this stock solution
(one-sixth of the total) is added 9.2 mg (0.061 mmol) of
phenoxyacetic acid, followed by 17.4 mg (0.09 mmol) of EDC. After
stirring for 20 hr, the reaction is processed as described above
for the isolation of intermediate IIIb, and the crude coupling
product is dissolved in 15 ml of 95% EtOH and hydrogenated at
atmospheric pressure (balloon reservoir) with 30 mg of 10%
Pd-on-carbon for 2 hr. After filtration through Celite and
concentration under reduced pressure, the crude product is purified
by preparative HPLC on a C18 column using a gradient of 0.1% aq.
NH.sub.4HCO.sub.3/CH.sub.3CN. Lyophilization affords 15 mg of
compound 8 (LC-MS, HPLC).
EXAMPLE 5
Synthesis of Other Compounds of the Present Invention
Compounds of Formulas 4 24 and 26 31 were prepared by employing the
methodology of Examples 3 and 4, substituting the appropriate
starting materials where necessary.
Given the guidance above, and combined with methods well known in
the art, one of skill in the art would be able to choose
appropriate starting materials in order to readily synthesize the
compounds of Formulas 4 24 and 26 31 as well as other compounds
within the scope of the present invention that are not specifically
exemplified herein.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing
description. Such modifications are intended to fall within the
scope of the appended claims. Various publications are cited
herein, the disclosures of which are incorporated by reference in
their entireties.
* * * * *